Antigen-specific mucosal immune responses, such as secretory IgA production and mucosal cytotoxic T lymphocyte (CTL) responses, may play an important role in host protection against infectious agents that infect via the mucosal surfaces of the host, including human immunodeficiency virus (HIV), herpes simplex virus (HSV), respiratory syncytial virus (RSV), Bordetella pertussis, and Salmonella typhimurium. See Ogra, in Mucosal Vaccines. p. 3 (1996).
A major obstacle in the path of developing effective mucosal vaccines is the identification of a safe and effective mucosal adjuvant. Indeed, mucosal immunization with soluble protein or peptide immunogens in the absence of a mucosal adjuvant tends to induce a state of antigen-specific immunological tolerance known as oral tolerance, or more appropriately, mucosally-induced tolerance. See e.g., Mowat, in Handbook of Mucosal Immunology. p. 185 (1994); Husby et al., Journal of Immunology 152:4663 (1994); Staines et al., Clinical & Experimental Immunology 103:368 (1996).
In contrast, mucosal immunization with soluble protein antigens or peptides co-administered with a mucosal adjuvant such as cholera toxin (CT), pertussis toxin (PT), or heat-labile toxin (LT) may induce potent systemic and mucosal, humoral and cell-mediated immune responses. Indeed, the most potent and best-studied mucosal adjuvant is cholera toxin (CT), Elson et al., in Handbook of Mucosal Immunology, p. 391 (1994). However, CT is likely unsafe for use as a mucosal adjuvant in humans because as little as 5 micrograms (.mu.g) of CT causes massive diarrhea when intragastrically administered to human volunteers. Levine et al., Microbiological Reviews 47:510 (1983). Moreover, in some cases the use of CT as a mucosal adjuvant in research animals has been associated with the production of antigen-specific IgE responses and lethal anaphylactic reactions. See e.g., Snider et al., J Immunol 153:647 (1994); Marinaro et al., J Immunol 155:4621 (1995).
To repress the toxicity associated with toxin adjuvants, mutant CT, LT, and PT molecules have been produced that exhibit reduced or undetectable toxic activity while maintaining mucosal adjuvant activity. O'Hagan, Journal of Pharmacy and Pharmacology 49:1 (1997). Although these molecules possess potent adjuvant activity with reduced toxicity, they maintain immunogenic properties when administered to experimental animals. See e.g., Douce et al., Infection & Immunity 65:2821 (1997). Thus, the immunogenicity of these mutant toxin molecules also prevents their widespread and repeated use as mucosal adjuvants in that pre-existing immunity to CT reduces their adjuvant activity. Wu et al., Vaccine 12:215 (1994).
PCT Publication No. WO 91/01143 to Pillai et al. describes interleukin (IL)-containing vaccine compositions which comprise a mixture of antigen and an adjuvant amount of an IL adsorbed onto a mineral in suspension, and a preservative. The mineral is described as preferably being alum. Alum is described as stabilizing the biological activity of the IL. Exemplary IL's includes IL-1.alpha., IL-1.beta., IL-2, IL-3, IL-4, IL-5, IL-6 and IL-7. It is noted that in the absence of alum, IL's have short half lives. Carbohydrate-containing units found uniquely on cancer cells or found associated with allergens are particularly described antigens. However, the problems associated with oral or mucosally-induced tolerance of antigens are not addressed.
U.S. Pat. No. 5.334.379 issued to Pillai et al. on Aug. 2, 1994 describes cytokine and hormone carriers for conjugate vaccines. The antigens described in this patent are bound or genetically fused with a cytokine, lymphokine, hormone or growth factor having immunomodulating activity. Particularly contemplated antigens include the carbohydrate-containing antigens often present in bacteria. Exemplary cytokines, lymphokines and hormones include IL-1.alpha., IL-1.beta., IL-2, prolactin, EGF, TGF, GM-CSF, GCSF, IGF-1, somatotropin, or insulin. The conjugates may be prepared by any of the known complex, biologically compatible methods for coupling carbohydrate-containing antigens or other antigens to carriers. Covalent coupling is described as a preferred method. Indeed, Examples 1 and 2 describe the elaborate reactions required to conjugate or bind the antigens to the cytokine, lymphokine, hormone or growth factor. However, the problems associated with oral or mucosally-induced tolerance of antigens are not addressed.
A recent review article by Kramer et al. entitled "Cytokine Mediated Effects in Mucosal Immunology" in Immunology & Cell Biology 73:389 (1995) discusses the role of IL-5, IL-6, and TGF-.beta. in the induction of mucosal IgA responses. Particularly, this paper discusses published results from experiments done in mice lacking a functional IL-5 or IL-6 gene. It also discusses papers which describe the co-expression of IL-5 or IL-6 with vaccine antigen in a live vaccinia virus, and which describe that the co-expression of IL-5 or IL-6 enhances mucosal IgA responses. However, the article then suggests that complex delivery methods will be required to deliver the cytokines to the mucosa.
Another recent review article by O'Hagan entitled "Recent Advances in Vaccine Adjuvants for Systemic and Mucosal Adjuvants" in the Journal of Pharmacy and Pharmacology 49:1 (1997) discusses the state of the use of adjuvants for systemic and mucosal administration. This review article discusses a number of different adjuvants for use with mucosally administered vaccines including particulates (i.e. microspheres), oil-in-water emulsions, and mutated forms of heat-labile enterotoxin (LT) and cholera toxin (CT). But, this article does not mention the use of cytokines as mucosal vaccine adjuvants.
A journal article by Lin et al. entitled "Present Status of the Use of Cytokines as Adjuvants with Vaccines to Protect Against Infectious Diseases" in Clinical Infectious Diseases 21:1439 (1995) discusses the use of select cytokines (IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, and IL-12; tumor necrosis factor (TNF); interferon; and GM-CSF as adjuvants. But, the use of cytokines as mucosal vaccine adjuvants is not suggested in this article.
A journal article by Nash et al. entitled "Recombinant Cytokines as Immunological Adjuvants", Immunology and Cell Biology 71:367 (1993) discusses the use of recombinant ovine IL-2, IL-1.alpha. and tumor necrosis factor-.alpha. (TNF-.alpha.) as adjuvants. The formulation of IL-1.alpha. with aluminum hydroxide (alum) is mentioned for use as an adjuvant capable of enhancing secondary humoral responses. But, there is no suggestion of mucosal administration of IL-1.alpha..
Despite the substantially non-toxic and biologically active nature of cytokines like the interleukins, the recognized uses of such molecules as adjuvants require complex conjugation or formulation techniques. Moreover, even in view of the potential benefits of mucosal immunization, a solution to the oral tolerance problem described above has yet to be identified. Thus, a safe, effective and easily formulated mucosal adjuvant is needed to aid the development of effective mucosal vaccines.